In nonalcoholic fatty liver disease (NAFLD) the cellular signals that initiate hepatocyte lipid accumulation and progression from benign steatosis to hepatocyte injury and chronic liver disease are unknown. Both experimental and human NAFLD are associated with hepatic oxidative stress along with overexpression of prooxidant cytochrome P450 (CYP) enzymes. Our general hypothesis has been that oxidative stress in this disease alters cell signaling cascades that promote the development of hepatocyte injury. Investigations over the past funding period have defined effects of oxidative stress on mitogen-activated protein kinases (MARK) that regulate hepatocyte injury and death. Other studies have demonstrated that oxidative stress generated by CYP2E1 overexpression alters hepatic metabolism including insulin sensitivity and lipid accumulation. Based on these studies and additional preliminary data, our central hypothesis is that overactivation of MAPK signaling by chronic oxidative stress is a critical mechanism in the development of both hepatic steatosis and injury. We propose to test this hypothesis with studies in hepatocyte and in vivo models of steatosis and oxidative stress that are contained in four specific aims. First, we will test the hypothesis that Bim down regulation is the mechanism by which CYP2E1-overexpressing hepatocytes resist death from oxidant stress. Second, we will test the hypothesis that CYP2E1-overexpressing hepatocytes are susceptible to proapoptotic JNK/AP- 1 overactivation because of redox-dependent phosphatase inhibition. Third, we will test the hypothesis that insulin resistance mediated by oxidant-induced JNK signaling promotes hepatocyte steatosis. Fourth, we will test the hypothesis that oxidative stress in the setting of lipid accumulation stimulates JNK mediated cell death from endoplasmic reticulum stress. The ultimate goal of these investigations is to better understand the basic cellular mechanisms that lead to the development of steatosis and steatohepatitis in order to design new strategies to prevent and treat human NAFLD. Relevance to public health: NAFLD is a very prevalent liver disease which has no known treatment. Attempts to understand the mechanisms of lipid accumulation and liver injury in this disease are important to the development of new strategies for the prevention and treatment of NAFLD.